Method of detaching micro-flora and compositions therefor

ABSTRACT

The invention relates to a method of controlling microorganisms adhered to surfaces in biological material, by administering substances which alter the water-structure binding capacity of surfaces and phase-boundaries. 
     To remove micro-organisms from the surfaces, organic and inorganic salts are used in combination with organic polymers, preferably biopolymers, with positively and/or negatively charged groups such as amino groups and carboxyl groups. 
     The invention also relates to compositions and preparations for performing the method, e.g. ointments, pastes and washing liquids containing the above-mentioned substances, as well as toothpaste containing one or more organic or inorganic salts together with the above-mentioned polymers.

The present invention relates to a method of greatly increasing thedetachment of micro-flora adhered to various types of boundarysurfaces/inter-phases by the addition of various combinations ofpolymers, preferably biopolymers, to treatment solutions. The inventionalso relates to preparations for achieving the above-mentioned result.

In our earlier patent application (Pat. app. No. 8206250-6) we were ableto establish that bacteria irreversibly adhered to various boundarysurfaces can be made to detach themselves to a greater or lesser extentsimply due to the action of concentrated salt solutions.

This is illustrated by, but not limited to the following experiments ina model system.

A number of glass tubes, all the same size, were immersed in asuspension of bacteria marked with a radioactive isotope (tritium) andthe bacteria were allowed to adhere over a period of 30 minutes. Theglass tubes were then removed one by one and rinsed by repeated dipping(30 times) in a buffer solution in order to remove reversibly boundbacteria. Only the irreversibly bound bacteria then remained on theglass tube. The glass tube was then immersed in a salt solution for 5minutes. After rinsing, the quantity of bacteria adhered to the tube wasdetermined by measuring the radiation in a liquid scintillator.

A comparison of the various glass tubes which had been immersed in saltsolution of various concentrations, with control tubes which hadundergone the same treatment with the exception of immersion in the saltsolution, enabled assessment of the effect of the salt solution on theirreversibly bound bacteria. The table below and the accompanyingfigures show the effects of the various salts on detachment of theirreversibly adhered bacteria, expressed as a percentage of the quantityof irreversibly adhered bacteria before the salt treatment. Theconcentrations of the solutions are in multiples of a specific criticalconcentration (C) of the basic solution.

Of the salts tested in this series of experiments, magnesium sulphate(MgSO₄) gave the strongest detaching effect. However, magnesium chloride(MgCl₂) and ammonium acetate (NH₄ Ac) solutions also gave good results,whereas ammonium sulphate ((NH₄)₂ SO₄) solutions gave a slightlydifferent pattern.

Several other compounds and salts have been tested besides thosementioned in this series of experiments, and the results were similar.

TABLE

Detachment percentage of irreversibly adhered bacteria after treatment.

The number of bacteria irreversibly adhered to the glass tube was3.0×10⁶ before treatment.

Salt concentration in multiples of basic solution C

    ______________________________________                                        Salts    0.05 × C                                                                           0.1 × C                                                                          0.2 × C                                                                          0.5 × C                           ______________________________________                                        MgSO.sub.4                                                                             41         54       28       28                                      MgCl.sub.2                                                                             20         40       21       25                                      NH.sub.4 Acetate                                                                       8.5        41       17       24                                      (NH.sub.4).sub.2 SO.sub.4                                                              31         36       44       28                                      ______________________________________                                    

The detachment of microorganisms as a result of exposure to solutions ofsalts and/or polymers can be seen in the attached drawings, in which:

FIG. 1 shows the detachment as a percentage of Streptococcus mutansbacteria bound irreversibly to glass, in relation to several salts atvarious concentrations.

FIG. 2 shows detachment as a percentage of Streptococcus mutans bacteriabound irreversibly to hydroxyl apatite, in relation to magnesium sulfateat various concentrations.

FIG. 3 shows detachment as a percentage of Streptococcus mutans bacteriabound irreversibly to glass, in relation to a combination of magnesiumsulfate at various concentrations, with various polymers at 1% byweight.

FIG. 4 shows detachment as a percentage of Streptococcus mutans boundirreversibly to glass, in relation to magnesium sulfate at variousconcentrations, together with combinations of two polymers.

FIG. 1 shows the detachment of irreversibly adhered bacteria aftertreatment with salt solutions of various concentrations. The saltconcentration is expressed in multiples of the basic solution C. Thenumber of irreversibly adhered bacteria was ca. 3.0×10⁶ per glass tubebefore treatment.

The curves are designated as follows:

MgSO₄ MgCl₂ NH₄ Acetate (NH₄)₂ SO₄

The detachment effect can also be seen in the following non-limitingexperiments and model systems.

1. A culture of radioactively marked bacteria was attached to hydroxylapatite beads, either coated or not with parotis saliva (saliva from thesalivary gland). The reversibly bound bacteria were then removed byrinsing with a buffer solution so that only irreversibly bound bacteriaremained on the hydroxyl apatite beads. These bacteria-coated hydroxylapatite beads were then exposed to various salt solutions of varyingconcentration and with the addition of biopolymers. After rinsing, thequantity of bacteria remaining was then determined by measuring theradiation remaining on the hydroxyl apatite beads in a liquidscintillator.

A comparison of the bacteria from the same quantity of hydroxyl apatitewhich had undergone the same treatment without having been exposed tosalt solution or biopolymers enabled assessment of the detachmenteffect. The result of a number of such detachment experiments onhydroxyl apatite can be found in FIG. 2.

The curves in FIG. 2 are designated as follows:

hydroxyl apatite beads

hydroxyl apatite beads coated in saliva

hydroxyl apatite beads coated in saliva and with the addition of 1%aminated starch (CATO 160) to the magnesium sulfate solution.

It can be seen here that the detachment effect of magnesium sulphate onsaliva-coated hydroxyl apatite is in the same order of magnitude asdescribed in the example performed earlier. Furthermore, the addition ofone (1) biopolymer (aminated starch) has substantially no influence onthe maximal detachment.

2. Using the model system with glass tubes described above, furtherexperiments were performed to determine the detachment effect ofmagnesium sulphate solutions with the addition of biopolymers. Theresults obtained are shown in FIG. 3.

The curves in FIG. 3 are designated as follows:

carboxylmethylcellulose 1%

xanthane 1%

aminated starch K 68 1%

aminated starch CATO 160 1%

It can be seen here that the detachment effect is not essentiallyimproved by the presence of one (1) of the biopolymers mentioned.

With higher concentrations of magnesium sulphate, the addition of two ofthe polymers produces a more pronounced detachment effect than with onlymagnesium sulphate.

Further detachment experiments were carried out using glass tubes asmodel system. The detachment effect of magnesium sulphate solutions withthe addition of two (2) biopolymers to the detachment solution wasinvestigated. Quite surprisingly it was found that several of thebiopolymer combinations gave considerably improved detachment effects.The result of these experiments can be seen in FIG. 4. It can be seenhere that all the polymer combinations used produce quite a differenteffect pattern from that obtained previously. It can also be seen thatxanthane in combination with other polymers produces a stronglyincreased detachment effect. The maximum detachment was approximately90%.

The curves in FIG. 4 are designated as follows:

xanthane 0.5% and aminated starch K 68 0.5%

xanthane 0.5% and aminated starch CATO 160 0.5%

xanthane 0.5% and carboxmethylcelluose 0.5%

carboxymethylcellulose 0.5% and aminated starch CATO 160 0.5%

aminated starch k 68 0.5% and CATO 160 0.5%

cellulose 0.5% and aminated starch K 68 0.5%

The method and preparation of the present invention can be utilized forthe removal of micro-organisms from various types of boundarysurfaces/inter-phases, e.g. in toothpaste or mouthwashes and incleansing liquids for wounds.

Besides the biopolymers mentioned above and in conjunction with thefigures, other biopolymers and other organic polymers may also be used.The polymers should preferably contain one positively and/or onenegativey charged group, e.g. one amino group and/or one carboxyl group.

We claim:
 1. A method for increasing detachment of microflora adhered tosurfaces and interfaces, comprising administering thereto a solutioncomprising;(a) at least one water soluble organic or inorganic saltselected from the group consisting of lithium, magnesium, aluminum,ammonium, beryllium, and calcium salts, and (b) at least two polymergroups selected from the group consisting of positively charged groups,negatively charged groups, amine groups and carboxyl groups.
 2. A methodaccording to claim 1, wherein said polymer groups are positively chargedgroups and negatively charged groups.
 3. A method according to claim 1,wherein said polymer groups are amine groups and carboxyl groups.
 4. Amethod according to claim 1, wherein the concentration of said at leastone salt is at least about 0.15 molar.
 5. A method for increasingdetachment of microflora adhered to surfaces and interfaces, comprisingadministering thereto a solution comprising:(a) at least one watersoluble organic or inorganic salt selected from the group consising oflithium, magnesium, aluminum, amonium, beryllium, and calcium salts, and(b) at least two polymer groups selected from the group consisting ofpositively charged groups, negatively charged groups, amine groups andcarboxyl groups, said polymer groups being supplied by including in saidsolution at least two polymers selected from the group consisting ofxanthan, aminated starch and carboxymethylcellulose.
 6. A methodaccording to claim 5, wherein said polymers are xanthane and aminatedstarch.
 7. A method according to claim 5, wherein said polymers arexanthane and carboxymethylcellulose.
 8. A method according to claim 5,wherein said polymers are carboxymethylcellulose and aminated starch. 9.A method for increasing detachment of microflora adhered to surfaces andinterfaces, comprising administering thereto a solution comprising:(a)at least one water soluble organic or inorganic salt selected from thegroup consisting of lithium, magnesium, aluminum, ammonium, beryllium,and calcium salts, and (b) at least two polymer groups selected from thegroup consisting of positively charged groups, negatively chargedgroups, amine groups and carboxyl groups, wherein said polymer groupsinclude positively charged groups and negatively charged groups suppliedby a single polymer.
 10. A composition for increasing detachment ofmicroorganisms adhered to surfaces and interfaces, said compositionbeing a solution consisting essentially of:(a) at least one watersoluble organic or inorganic salt selected from the group consisting oflithium, magnesium, aluminum, ammonium, beryllium and calcium salts, theconcentration of said at least one salt being at least about 0.15 molar,(b) at least two polymer groups selected from the group consisting ofpositively charged groups, negatively charged groups, amine groups andcarboxyl groups, and (c) water.
 11. Composition according to claim 10,wherein said solution contains, as polymer groups, amine groups andcarboxyl groups.
 12. A composition for increasing detachment ofmicroorganisms adhered to surfaces and interfaces, comprising a solutioncomprising:(a) at least one water soluble organic or inorganic saltselected from the group consisting of lithium, magnesium, aluminum,ammonium, beryllium and calcium salts, the concentration of said atleast one salt being at least about 0.15 molar, and (b) at least twopolymer groups selected from the group consisting of positively chargedgroups, negatively charged groups, amine groups and carboxyl groups,wherein said polymer groups are supplied by at least two polymersselected from the group consisting of xanthane, aminated starch andcarboxylmethylcellulose.
 13. Composition according to claim 12,comprising xanthane, together with aminated starch orcarboxymethylcellulose.
 14. A composition for increasing detachment ofmicroorganisms adhered to surfaces and interfaces, comprising a solutioncomprising:(a) at least one water soluble organic or inorganic saltselected from the group consisting of lithium, aluminum, ammonium,beryllium and calcium salts, the concentration of said at least one saltbeing at least about 0.5 molar, and (b) at least two polymer groupsselected from the group consisting of positively charged groups,negatively charged groups, amine groups and carboxyl groups.
 15. Acomposition according to claim 14, wherein said solution contains aspolymer groups, amine groups and carboxyl groups.